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Full text of "The germ-cells and the process of fertilization in the crab Menippe mercenaria .."

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THE GERM-CELLS AHD THE PROCESS OF FERTILIZATION 

In tha 
CRAB MEHIPPE MERGEHARIA 

Raymond Binford. 



A DISSERTATION 

Submitted to the Board of University Studies 
of the Johns Hopkins University in conformity 
with the Requirements for the Degree of Doc- 
tor of Philosophy. 



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THE GERM-CEILS AUD THE PROCESS 05* FERTILIZATION 

in the 
CRAB IvIEUIPPE IvIERCElTARIA. 



Contents . 

Page. 

I . Introduction "b-c 

II. Spermat ogene si s : 

1. The testis 1 

2. Methods 1 

3. The testicular tuhules 2-4 

4. General statement of spermatogenesis 4-5 

5. The spermatogonial mitoses 5-6 

6. Maturation mitoses 6-9 

7. The transformation of the spermatid into 

the sperm 9-14 

8. The sperm 14-15 

9. Sperms in the deferent duct ..15 

lu. Discussion 15-17 

III. Copulation 17-18 

lY. Spawning hahits 18-19 

V. The reproductive organs of the female 19-21 

VI. The "behavior of the sperms: 

1. Methods of study 21-22 

2. Changes in the nuclear cup 23 

3 . Change s in the capsule 23-25 

4. Changes in the central "body 25-27 

6. The dynamics of eversion 27-32 

6. The effect of reagents on everted sperms. .. .32-33 

VII. The entrance of the sperm 33-36 

VIII. Fertilization 36-39 

iZ. Discussion 39-42 

Z. Summary 42-44 



IlITRODUCTIOU. 

In spite of the extensive researches iat© the spermato- 
genesis of the Decapods, the use of the peculiar structures 
found in the sperms of these animals is still an unsolved 
prol)lem. This is due to the fact that the entrance of the 
sperm into the egg has never "been reported. While studying 
the hat its and structure of Menippe mercenaria, a large 
edible crah found along the southern part of the Atlantic 
coast of the United. States, I had the good fortune to obtain 
material which shows the essential features of this process. 
In order to show which parts of the seminal cell are involved 
in the process of fertilization, the genesis of the sperm 
and the formation of the pronucleus in the fertilization of 
the egg, as well as the entrance of the sperm, are here de- 
scribed. The history of the male cell from its origin in 
the epithelium of the wall of a tubule of the testis to its 
association with the female nucleus in the center of the egg, 
is here presented. 

The study of the life-history of Menippe mercenaria, 
which led to the discoveries presented in this paper, was 
undertaken at the suggestion of Prof. E. A. Andrews, and at 
every step in the progress of this work I have received his 
kind advice and helpful criticism. I am also greatly indebt- 
ed to the Hon. Geo. M. Bowers, United States Commissioner of 
Fish and Fisheries, for the privilege of working in the 
Marine Biological Laboratory at Beaufort, II. C., and for the 



lilDeral help extended to me in carrying on ray researches there. 
My thanks are also due to Mr. H. D. Aller, Director of the 
Laboratory, for his ready cooperation in placing at my dis- 
posal the conveniences necessary for carrying forward my 
work. 



SPERMATOGENESIS. 

1. The Testis. 

The testis of Menlppe Is a large paired organ lying Just 
underneath the dorsal wall of the carapace. The Inner ends 
of the right and left portions lie close together Just anter- 
ior to the heart and from here diverge anteriorly and later- 
ally to the outer edge of the carapace. It is composed of 
relatively long and complexly folded tubules which vary in 
diameter from 0.14 to 0.33 mm. The deferent duct, one on 
each side, leads from the testis to the base of the last 
thoracic leg. It is extensively convoluted so as to form 
two large masses, one lateral to the posterior part of the 
testis and the other beneath the posterior part of the heart. 
The deferent duct is lined with a layer of columnar epithelium 
which secrets the substance that forms the walls of the 
spermatophores . 

2. Methods . 

Pieces of the testis, obtained by cutting across the 
organ, were fixed in Worcester's fluid. This is a saturated 
solution of sublimate in 10^ formalin. Other fixing fluids 
were used but did not give as satisfactory results. The sec- 
tions were cut Iji to lOjx thick. The stains used were thionln 
and eosin, safranin and LichtgrUn, iron-haeraatoxylln, and 
Delafleld's haematoxylln. 



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3. The TeBtiottlar Tatules. 

The walls of the tubules of the testis are thin and con- 
tain flattened nuclei. Figures 1, 2, 3 and 4 are drawings of 
transverse sections of the tuhules and show different stages 
In the development of the seminal elements. In Pig. 4 the 
tubule is seen to be made up of three regions: The smallest 
one which bordars the more sharply curved side at the bottom 
of the drawing contains mature sperms. Next to this and fill- 
ing the central region is a space filled with sperms nearly- 
mature. The third region, which forms the crescent shaped por- 
tion on the tipper side, contains spermatocytes in the early 
propase of the first maturation division. There are more or 
less definite layers of epithelial cells between the different 
regions of the tubule. The outer wall and sometimes these 
inner partitions which border the regions containing mature 
sperms, become thick and columnar in structure (Pigs. 3 and 
4). 

Hot only do the seminal elements in these separate paral- 
lel cavities of the tubule differ in th« stages of their devel- 
opment but in the same cavity the elements at one end of the 
tubule are further along in their development than those at 
the other end. Thus In one end of a cavity the cells may be 
In the early prophase of the division of the spermatocytes 
of the first order while in the other end they have reached 
the spermatid stage. All the stages in the transformation 
of a spermatid into a sperm may be found in passing from one 
end of a tubule to the other. 



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At the center of the upper border of Fig. 4, p.s., there 
is one cell with a large nucleus. This is one of a single 
row of cells along the side of the tubule which may be called 
the prlDBtry sperraatogonial cells since they, by division, 
give rise to a new lot of spermatogonia. Hear the top of Fig. 

3, p«s., we find a similar cell. The cells forming the cres- 
cent-shaped region are In this case not so far advanced as in J*'ig. 

4. Figure 1 represents a tubule the largest portion of which 
is filled with spermatids which have already entered upon their 
transformation into sperms. In the upper portion of the draw- 
ing we have an early stage in the foimation of a new batch of 
spermatogonia. There are four large spermatogonial nuclei 
surrounded by many epithelial nuclei and a considerable amount 
of cytoplasm. Delicate cell walls cutting O'.t the cytoplasm 
that belongs to each spermatogonial cell can sometimes be made 
out at this stage. A later stage in the multiplication of 
these cells is shown in Fig. 2. A large nucleus, p.s,, near 
the middle of the convex border of the spermatogonial mass, 
doubtless marks the position of the row of primary spermatogon- 
ial cells which will persist unmodified to form, at a later 
period another lot of spermatogonia. The largest cavity of 
this tubule contains spermatids well advanced in their trans- 
formation into sperms. They fill the cavity but only a por- 
tion of those seen in the cross-section are represented in the 
drawing. In Fig. 3 the mass of spermatogonial nuclei is still 
further enlarged. Indeed most of thorn have probably reached 
the spermatocyte stage. The spermatocytes in the early prophase 
of the first maturation division are shown in Fig. 4. By put- 



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ting these observations together we may determine the approxi- 
mate order of events In the genesis of the sperms. 

4. A General Statement of Spermatogenesis * 

There persists along one side of the tuhnle a single row 
of cells with large nuclei, the division of which give rise to 
the spermatogonia. The latter multiply irregularly to form a 
large mass which in transverse section has the shape of a 
crescent. At first cell walls can "be made out, hut later the 
nuclei seem to lie in an undivided mass of cytoplasm. Grad- 
ually the division of these nuclei ceases and a spireme is 
formed within each of them. The division up to this time has 
taken place without the formation of any spireme structure. 
The appearance of the latter is the first indication that the 
cells have reached the spermatocyte stage. After the spireme 
has been formed the nuclei pass into synapsis which lasts for 
a comparatively long time, so that all the nuoell in a con- 
siderable portion of a tubule will be found in this stage at 
the same time. After synapsis cell walls are formed in the 
cytoplasm which persist up to the anaphase of the first ma- 
turation division. In the nucleus the chromosomes are form- 
ed and the maturation division follow one another in quick 
succession. They begin at one end of the tubule and pass along 
it like a wave so that the spindle-figures are found in only 
a small section of the tubule at any given time. While these 
events are taking place within the tubule the cells in the 
wall of the latter multiply so that the wall becomes consider- 
ably thickened. The primary spermatogonia! cells also divide 



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to start a new group of spermatogonia. Between these cells 
and the spermatocytes there Is always a layer of epithelial 
cells which persist to form the partitions "between the two 
successive hatches of seminal elements. The mass of sperma- 
togonlal nuclei remains small until the spermatids are well 
advanced In their transformation into sperms. 

As the mass of spermatogonia Increases the developing 
sperms are crowded more and more to one side of the tuhule. 
These sperms reach their mature state hefore the second batch 
enter synapsis. The epithelial cells surrounding the mature 
sperms, we may suppose, secret a fluid which together with the 
increasing mass of spermatogonia press the mature sperms out 
of the tubule. This process is not completed, however, before 
a third batch is formed or even a fourth started. 

5. Spermatogonial Mitoses . 

In the resting spermatogonia! nucleus the chromatin is 
arranged in a loose net-work with enlargements at various 
places tPig. 5). This net, for the most part, lies just in- 
side the nuclear membrane, the central part of the nucleus 
containing almost no staining material. The behavior of the 
chromatin during the prophase of Bltosis is as follows; the 
knots of chromatin become enlarged and more regular in out- 
line while the connecting threads become smaller and disap- 
pear. The chromatin finally assumes the form of a large num- 
ber of paired spheres (Pig. 6i. An effort was made to count 
these spheres and numbers were obtained as follows: 61, 55, 
67, 58, 6E, 62, 68 and 80. One may not however place very 



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muoh dependence in these numbers for soiirie of the spheres are 
always somewhat aggregated In one or two places so that they 
oan not be definitely distinguished. These chromosomes at 
first lie in the outer part of the nucleus just inside the 
nuclear membrane, but are later massed in the center, from 
which condition they move to their positions in the equitorial 
plate. Figure 7 is an optical section of the nucleus showing 
the peripheral arrangement of the chromosomes, m the meta- 
phase and anaphase of the mitosis the members of each pair 
are separated from each other and pass to opposite poles of 
the spindle iPig. 8>. These divisions of the spermatogonial 
nuclei do not occur simultaneously throughout the mass, but 
singly here and there amongst the nuclei. The spermatogonia 
become smaller as they become more numerous. 

6. Maturation Mitoses . 

Finally the spermatogonial divisions cease and the nuclei 
prepare for the reduction divisions. The quantity of chromatin 
seems to increase and the spireme makes its appearance. At 
first it is very long and slender and complexly folded all 
through the nucleus. The iron-haematoxylin stain can be con- 
trolled so that the spireme has the appearance of a brown 
thread with granules distributed irregularly along it iFlg.9). 
The diameter of the granules is slightly greater than that of 
the thread between the granules. The spireme now becomes 
shorter and thicker and is finally massed at one side of the 
nucleus in the condition of synapsis (Fig. 10). This stage 
persists for a comparatively long period. The spermatocytes 



enter sTnapsls Irregularly, in a sort of one -at-a- time fashion 
but they tarry here tmtll all of the cells In the greater part 
of the tuhule have reached this stage, then the nuclei of a 
given portion all proceed to the open spireme stage, shown in 
Pigs. 11 and IE. These figures show only the chromatin which 
lies on the side from which the nucleus was observed. The 
chromatic material is again arranged in the peripheral portion 
of the nucleus and is segregated Into the chromosomes which 
become somewhat massed In the center of the nucleus. The 
spindle next makes its appearance (Fig. 13 J and the chromosomes 
are drawn into the equatorial plate (Pig. 14). 

The mitotic figure represented in Pig. 15 shows the pos- 
sibility of a tri-polar division. Such a condition may have 
been brought about by the formation of one of the spindles of 
the second division before that of the first division was com- 
pleted. There is a small portion of the chromatin of this 
nucleus that is not Involved in the mitotic figure. This 
portion is shown at £., in Pig. 15a which Is a drawing of 
what was seen at a lower level than that shown in JTig. 16. 

The chromosomes in these nuclei are so small and so close- 
ly crowded together it is very difficult to determine their 
structure or their number. In one preparation, however, I 
obtained a ring-shaped appearance of the chromosomes lPig.l6). 
These forms were seen in the equatorial plate and also before 
the chromosomes had been arranged in the plate. In most of 
the preparations the chromosomes appear as mere granules. It 
may be that the ring shaped forms were produced by the fixing 



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reagent, which may have caused a ewelllng of the chromosomes. 
This result was not always obtained however hy the same re- 
agent . 

when de staining Is carried so far as to remove all the 
stain from the cytoplasm and the achromatic figure, the equa- 
torial plate may be shown to have a structure like that repre- 
sented in Pig. 16a. This was drawn from a section cut from 
the edge of the plate. Here it appears that the chromosoines 
are stretched as they are pulled apart. Strands of chromatin 
pulled out between the separating groups of chromosomes may 
be seen In the later stages of the anaphase. By more exten- 
sive destainlng we may obtain what appears to be only the 
cores of the chromosomes as shown in Pig. 16b. 

In Pigs. 17 to 22 various stages in the anaphase are rep- 
resented. The interzonal fibers and the mid-body axe very 
distinct In Pigs. 19 to 21. 

The second mitotic division follows very soon after the 
first. The chromosomes become somewhat separated and are 
then drawn together again Into the equatorial plate ready 
for the second division (Pigs. 23 to 25). Plgure 26 shows 
the beginning, and Pig. 27, the end of the anaphase. Here 
again the Interzonal fibers and the mid-body are distinctly 
seen and a portion of the cytoplasm is definitely associated 
with each daughter nucleus. The nucleus of the spermatid 
Is now organized and persists in a sort of resting condition 
for a comparatively long time. The centrosome may also be 
distinguished for a considerable time but later I was unable 



to recognize it (Figs. 28 to 32). A clear space surrounding 
the nucleus Is also seen in these figures. The spermatid as 
it appears In Pig. 32 rests for a conslderahle period hefore 
any change towards the formation of the sperm is observed. The 
boundaries "between the cytoplasm of the different cells dis- 
appear and the nuclei come to lie in a sort of Plasmodium. 

7. T he transformation of the spermatid into the sperm . 

In serial sections of a single tuhule we may trace every 
stage in the transformation of the spermatid into the sperm, 
and since the two ends of the series are in opposite ends of 
the tubule and the intermediate stages lie in serial order 
between these ends we may use the position of a seminal ele- 
ment in the tubule as a criterion for determining its relative 
stage in the course of development. The first evident step in 
the transformation of the spermatid is the appearance of 
vacuoles in the cytoplasm next to the nucleus. These are at 
first small but by coalescing they soon form a large, clear 
vacuole on one side of the nucleus (Figs. 3J5 to 38). Some- 
times it appears that the vacuole may have arisen by the nu- 
cleus settling to one side of the clear space surrounding it 
in Pig* 31. The nuclei, each with its accompanying vacuole, 
now lie in a common mass of cytoplasm. In the further develop- 
ment of these cells there are three parts that must be con- 
stantly borne in mind, viz., the nucleus, the vacuole (here- 
after called the capsule) and the cytoplasm. We shall take 
up certain stages in the differentiation of these three 



10 



parts, and consider their relation to each other. 

In Figs. 37 to 41 the shape of the nucleus may he some- 
T?hat modified by strains In the cytoplasm or by the crowding 
of the elements in the tubule. In these drawings there is 
no evidence of a granular or reticular structure, although 
such structure was made out in some preparations which were 
destained to a greater degree. In Fig. 37 It may be observed 
that the outer layer of the nucleus stains more densely than 
the inner portion. The nucleus in Fig. 38 contains a vacuole 
which does not take the stain. The cytoplasm surrounding the 
nucleus and capsule in Figs. 37 to 39 Is nearly uniform in 
appearance, with probably a tendency to be a little more deep- 
ly stained near the nucleus. In Figs. 40 and 41 there is a 
concentration of a portion of the cytoplasm on one side of the 
capsule and bordering the nucleus. This is finely alveolar 
and stains more deeply than the rest of the cytoplasm. It 
may be that this patch of cytoplasm is seen in an earlier 
stage In Fig. 36c. This portion of the cytoplasm crowds in 
between the nucleus and the capsule (Fig. 42). About this 
time the capsule begins to take a brownish color when stain- 
ed with iron-haematoxylin. 

The otigin and development of this portion of cytoplasm 
which appears on the side of the capsule and nucleus and 
wedges in between them is a striking feature In the develop- 
ment of the sperm. Its behavior is well brought out in Figs. 
45-48. In Fig. 45 we see this substance slipped in like a 
wedge between the nucleus and the capsule with a clear space 
between it and the nucleus. If the spermatid shown in this 



11 



figure were rotated to the right through 90** so as to hrlng 
the outer surface of the wedge of cytoplasm on the side to- 
ward the observer we would have the appearance presented In 
?lg. 46. If we should turn this through 180° so as to throw 
the wedge on the opposite side from the observer the spermatid 
would appear as in Pig* 47 where just the tips of the orescent 
shaped wedge are seen. The tips of this crescent progress on 
so'ound the capsule along the boundary line between the nucleus 
and the capsule. At the same time the thick side of the wedge 
Is reduced and the material Is distributed equally around this 
border-line to form a complete ring, which viewed from any 
lateral direction, has the appearance shown In Fig. 48. At 
first the substance of the wedge Is finely alveolar In appear- 
ance but by the time the ring Is completed It seems to be uni- 
form throughout and Is stained black with Iron-haematoxylln. 
It seems to be identical with the mitochondrial substance de- 
scribed by Koltzoff '06. 

After the mitochondrial ring is completed, the nucleus 
becomes widely separated from it and the capsule (Figs. 50 
to 52). This however Is not always the case. In two prepara- 
tions from which Figs. 33 to 35 and 37 to 43 were drawn, the 
nucleus remained fitted closely down on the capsule as shown 
in Fig. 43. As the two different conditions were obtained 
with the same fixing fluid it is hardly probable that the 
difference was caused by the fixing. The nucleus at this time 
looses the last trace of any granular or reticular structure 
and becomes uniform ih its staining reactions, and somewhat 
reduced in size. 



12 



About the time the mitochondrial mass begins to slip in 
between the nucleus and the capsule, one or two deeply stain- 
ing granules appear on the border line between the nucleus 
and the capsule (Figs. 44 to 48). Koltzoff '06 in his re- 
searches on the spermatogenesis in Galathea squamifera has 
identified these granules with the centrosome. In my prepara- 
tions of Menippe meroenaria I am able to distinguish the centro- 
some for some time after the second maturation division (Pigs. 
28 to 3E), but in the later resting period of the spermatid 
and in the stages during the origin of the capsule, I am unable 
to distinguish any granule that can with any certainty be iden- 
tified with the centrosome » I shall call the structure develop- 
ed from this granule, the central body . I am unable to follow 
the development of two distinct granules although two could 
some times be clearly distinguished as shown in ?ig. 44. Prob- 
ably only the outer one is concerned in the development which 
is here presented. 

This outer granule elongates (Fig. 47) and becones tubular 
(Figs. 50 to 56). There soon appears at the outer end a vesicle 
which Increases in size as the central body elongates. yiThile 
the vesicle is still small there appears in its outer wall a 
flattened granule which Is usually seen to be connected with 
the end of the central body by means of a fine strand as though 
it might have been derived from this body. As the central 
body Increases in length and the vesicle enlarges its outer 
wall approximates the outer wall of the capsule. The deeply 
staining substance in the outer wall of the vesicle now becomes 
connected with the wall of the capsule. (Pig. 56 to 58). A sec- 



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13 



ond vesicle now forms. (Pig. 59). These two vesicles "become 
transformed Into a tu"bule containing the central body. This 
tuhule will hereafter be spoken of, as the inner tubule . At 
its outer end a ring of darkly staining substance is found (Pig. 
60). This seems to have* been derived from the central body. At 
least a study of Pigs. 54 to 60 may well suggest such an inter- 
pretation. The central body finally becomes reduced in diameter 
and appears to be a solid rod. It is not stained by thionin, 
nor by safranin,but is readily stained with iron-haematoxylin. 

The inner tubule is stained green with safranin coimter 
stained with Lichtgriin; blue with thionin counter stained with 
eosin; and black with iron-haematoxylin. 

During this whole period the content of the capsule shows 
an increasing affinity for chromatin stains. It is colored 
brown with iron-haematoxylin. In some series a sort of ring 
shaped cloud appears in the capsular contents. At first it is 
near the outer wall but gradually it contracts towards the ves- 
icle at the end of the central body and finally settles in the 
wall of the tubule when that structure takes its final form. 
With Delafield's haematoxylin the contents of the capsule is 
readily stained and with safranin it takes a dull red color. 
In the early stages of development the content of the capsule 
is stained green when the preparation is treated with the 
safranin and Llchtgrun combination, but in the later stages 
the green is masked by the red. In stages represented in 
Pigs. 53 to 55 a sort of foam or alveolar structure can some- 
times be observed in this substance. 

While the capsule and the structures within it are assum- 



bevxret 



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14 



Ing their mature form the nncleus has heoome less densely 
stained and settles down upon the capsule like a cap. (Pigs. 
62 to 59). It becomes thin In the center so that Its final 
shape Is that of a cup with a rounded, thin hottom and a 
thickened rim. This thickened border fits upon the mltoohron- 
drlal ring so that In the mature sperm It is not possible to 
distinguish It from that ring. 

Protoplasmic rays or pseudopodla develop out from the 
rlm of the cup. I have been unable to determine whether they 
arise from the mitochondrial substance or from the' nucleus. 

8. T he Sperm . 

We may now consider the structure of the mature sperm. 
Figure 61 Is a drawing of a sperm taken from the seminal recep- 
tacle of the female and killed in the vapor of osmlc acid then 
stained with gold chloride after treatment with formic acid. 
We observe the nuclear cup, n.c. from which the pseudopodla, 
ps. arise. Inside the cup Is the spherical capsule, c. within 
which there is the capsular oavlty, c.c; and the inner tubule, 
l.t. with its cavity divided Into the inner tubular cavity, 
l.t.c. ; and the outer tubular cavity, o.o. Running through 
the Inner tubular cavity and through the wall of the inner end 
of the tubule to the bottom of the capsule we see the central 
body, c.b. Figure 62 was drawn from a live sperm in A% ENO3, 
and Pigs. 63 to 65 are from sperms mounted in the serum of 
the crab' s blood. Movements of the blood have bent the pseudo- 
podla of these sperms. Otherwise they have more nearly the 
natural shape and proportions than those shown in Pigs. 61 and 



10 



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15 



62. The diameter of the capsule of these sperms is about 
3.8^ and the pseud opodia are some times as much as 7^ long* 

9. Sperms in the deferent duct. 

The mature sperms pass from the tuhules of the testis in- 
to the deferent duct. The latter is a long extensively fold- 
ed tuhe lined with glandular epithelium. The sperms form a 
common mass when they enter this tube but the secretion form- 
ed by its lining flows in amongst them and separates them in- 
to groups. The secretion surrounding each group then hardens 
and so forms a membrane, so that finally there are an immense 
number of capsules containing the sperms. These capsules are 
known as spermatophores. In this condition the sperms are 
transferred to the seminal receptacle of the female crab. 

10. Summary and discussion . 

In this study of spermatogenesis in Menippe mercenaria 
the principal points brought out are as follows: 

1. There is a single row of cells which persists on one 
side of the testicular tubule and gives rise to successive 
hatches of spermatozoa. 

2. The spermatogonia divide without the formation of a 
spireme. The chromatin simply aggregates into chromosomes 
which are then gathered into an equatorial plate. 

3. The maturation divisions follow one another quickly. 
They are proceeded by spireme formation and a long period of 
synapsis. 



.flc..*-ei-v^f 



16 



4. There also seems to be a relatively long resting 
stage after the nucleus of the spermatid Is formed hefore the 
transformation Into the sperm heglns. 

6. In the transformation of the spermatid, three struc- 
tures must he considered, viz., the nucleus, the capsule and 
the mitochondrial ring. 

^. The nucleus becomes uniform In consistency, reduced 
In size and cup-shaped. 

7^» The capsule arises In the cytoplasm as a clear 
vacuole which may be stained V7lth Llohtgrun. Its content is 
gradually changed to have a greater affinity for chromatin. 

8,. Prom a granule on the proximal side of the capsule 
the central body develops Into the capsule. At the distal 
end of this body a vesicle arises, which is changed into the 
inner tubule. 

9. The mitochondrial substance is segregated from the 
cytoplasm and deposited as a ring between the nucleus and 
the capsule. 

Some of the theoretical questions connected with the 
development and structure of the sperms of the decapods will 
be taken up at the end of this article. At this point I wish 
to say that the above description Is in agreement with the 
principal observations made by Grobben '78, Gllson '86, Saba- 
tler '93, Brandes '97 and Koltzoff '06. These authors have 
all seen the same general structures and transformations. 
They all describe a nucleus which during development Is modi- 
fied in its staining reactions, reduced in size and often 
flattened or otherwise changed in its shape. They do not dis- 



17 



agree as to which part of the cell is the nucleus. They like- 
wise desorihe a vesicle which arises in the cytoplasm either 
against the nucleus or close to it, and mention the substance 
of cytoplasmic origin which appears hetween the nucleus and 
the vesicle. Most of them see a structure like the central 
body and describe the inner tubule. There are many variations 
in the detail of the development of these last two structures 
and different species seem to differ widely in this respect. 
There is mxxch disagreement concerning the destiny of the nu- 
cleus and the origin and nature of the substance in the cap- 
sule. These points of disagreement do not however affect the 
statements I have made concerning the structure of the mature 
sperm. It is with this structure that we have to do in the 
further course of the present investigation. 

III. Copulation . 

We come now to the question of the transfer of the sperm- 
atophores from the body of the male to that of the female, 
from the deferent duct to the seminal receptacle. We there- 
fore turn our attention from the sperm itself to some of the 
habits of these crabs. Menippe mercenaria lives in crevices 
under or between the rocks or in burrows which it digs in 
the mud along the shore a little below low water line. Usual- 
ly one crab is found in each burrow, but occasionally, and 
even frequently in the month of August, a male crab will be 
found guarding a hole in which there is a female. Sometimes 
the female thus found has a soft shell. If its shell is 



18 



hard It molts within a few days after helng hronght Into cap- 
tivity. On Aug. 17, a female with a soft shell and a male 
crab which had been taken from the same hole about noon were 
placed together In a compartment of a floating cage. At 
5:45 P.M. they were observed to be copulating. On being dis- 
turbed they separated. Their behavior was then observed 
while copulation was resumed. The most significant point 
with regard to this behavior was the apparent care with which 
the male acted in order to inflict no injury upon the soft, 
delicate shell of the female. 

During copulation the spermatophores are transferred from 
the deferent duct to the portion of the seminal receptacle 
that is lined with chitin, where they are deposited in a very 
compact mass. Here they remain until the next spawning of 
eggs. Only a portion of the sperms are used for the fertili- 
zation of any one batch of eggs. One crab kept by itself in 
a compartment of a floating cage for 69 days during the summer 
of 1911, spawned six times sind apparently all of the eggs 
in the six different batches of 500,000 to 1,000,000 eggs each, 
were fertilized and developed normally. 

IV. Spawning Habits . 

The spawning habits and the development of this crab will 
be discussed In a later paper. Here we shall present only 
such points as are necessary in order to make it clear how the 
stages in the entrance of the sperm and fertilization are ob- 
tained. 



jfilti^oX' 



19 



When a female is ready to lay a batch of eggs she assumes 
an upright position and holcis the abdomen out from her body 
so that it and the exopods of the abdominal appendages form 
a basket into which the eggs are run. They there become at- 
tached to the hairs of the andopods of the appendages and 
pass through the embryonic stages of their development, which 
requires from nine to thirteen days. The eggs then hatch and 
the larvae escape. The female then cleans off the egg-shells 
and their stalks from the hairs of the pleopods and after one 
day to three weeks she spawns again. Eight days is a very 
common length for the period between the hatching of one batch 
of eggs and the spawning of the next. With these facts in 
mind I made a large floating cage with fifty compartments and 
collected a large number of females with eggs and placed one 
in each compartment. After tjfie eggs of several of these had 
hatched so that there were some fifteen crabs without eggs I 
kept these under almost constant observation, day and night. 
When one assumed the position ready for spawning it was natur- 
ally supposed to contain eggs that were mature if they were 
not already fertilized. Before describing the process of 
fertilization we should consider briefly the structure of the 
genital organs of the female. 

V. TH3 RfiPRODUGTIVE ORGMS OF THE FEMALE. 

Figure 181 Is a diagramatic representation of the ovary 
and one seminal receptacle and oviduct of this crab. The 
ovary is an H shaped tube, the lumen of which opens directly 



80 



into the seminal receptacle at a point a little posterior to 
the cross connection of the H. The eggs are produced in the 
wall of this tuhe and when mature are set free in the lumen. 

The seminal receptacle is composed of two parts, a 
glandular portion (Pigs. 121 and 122, g.) into which the ovary 
opens and a portion lined with chitin (Figs. 121 and 122, c.) 
from which the oviduct leads to the third segment of the 
sternum. The speraatophores are stored in the latter division. 
The cavities of the two portions communicate through a large 
opening (Fig. 121,0.) in the chitinous lining. Just before 
the crab molts the glandular portion secretes a mass of 
gelatinous material which greatly distends it (Fig. 122) and 
the spermatophores are "by some means transferred to this part 
of the receptacle where they lie in the mass of jelly. This 
prevents them from "being lost at the time of molting when the 
chitinous lining is shed. Whether they are returned to this 
part of the receptacle after the molting has not been deter- 
mined. The glandular part of the receptacle is rapidly re- 
duced after the shell is shed, but I do not know what becomes 
of the secretion. During spawning the glandular portion is 
very much contracted (Fig. 121) so that it is little more than 
a tube connecting the ovary with the chitinous receptacle. 
There is one possibility which may be mentioned here and that 
is that the glandular receptacle may secret a semi-fluid sub- 
stance and then by contracting force the sperms Intoithe lumen 
of the ovary Just before spawning begins. Asni I shall show 
later the sperms are transferred to the ovary. This however 
is only a conjecture as to the method of the transfer. The 



i":s '. 97orf 



ibt^e^s 



21 



only time at which the receptacle is known to "be actively 
secreting a suhstance is Just "before molting and it may simply 
be a device for retaining the spermatophores at the molting 
period. 

If a crah that has Just hegun to lay its eggs is opened 
the lumen of the ovary and the oviduct will he found to he 
full of eggs. Some eggs were taken from the lumen of the 
ovary with a sterilized plpet and placed in filtered sea- 
water. Since these developed into emhryoes it is evident 
that fertilization takes place in the ovary. Sections were 
made of eggs taken from the lumen of the ovary and from the 
oviduct smd from these the phenomena of fertilization were 
observed, hut we shall return to this later. 

VI. THE BEHAVIOR OP THE SPERMS. 

The sperms of this crah sure so very minute, the eggs so 
relatively large and opaque, and the conditions for sperm en- 
trance so difficult to reproduce on the microscopic slide, 
I did not see the living sperm enter the egg. It is easy 
however to interpret the structures seen in sections of eggs 
taken at spawning time, after one has observed the behavior 
of the spermatozoa under certain experimental conditions. We 
shall proceed therefore to a description of this behavior. 

1. Methods of Study. 

Koltzoff '06, by his careful analysis of the effects on 
the sperm of solutions differing in osmotic pressure, has 



22 



Cleared up many of the mysteries of the decapod sperm. Accord- 
ing to his researches the sperms maintain their normal form 
in solutions of salts having the same osmotic pressure as 
sea-water. He also found that 5'^ KNO3. 2.8^ HaCl, 4.25^ UalTOg, 
18.5^ MgS04, 1% glycerine and 25.66/S sugar solutions are 
isotonic with sea-water. Solutions of these salts at a lower 
concentration cause a deformation of the sperms. 

For my studies solutions of KHO3, UaCl and BiaH03 were 
used. The sperms taken from the seminal receptacle and placed 
in solutions of these salts isotonic with sea-water would re- 
main many days without perceptahle change. When they were 
placed in weaker solutions of these salts transformations oc- 
curred. In studying these changes I proceeded as follows: 
Sperms from the seminal receptacle were placed in the serum 
of the crab's blood or in the isotonic solutions of KNO3, 
HaCl, or HaHOg. In these solutions they were transferred to 
the slide, covered and examined under the high power of the 
microscope. Then, by placing a weaker solution of one of the 
salts at the edge of the cover-glass and allowing it to dif- 
fuse underneath, a slow change in the form of the sperm was 
obtained. This change was thus followed in detail. It is to 
these changes that we will now turn our attention. 

By referring to Pig. 61 we may again call to our minds 
the normal condition of the mature sperm which consists of a 
chltinous capsule, set in a protoplasmic cup. The capsule 
contains a tubule with an inner and outer cavity and running 
through the inner cavity of the tubule is the central body, 
the proximal end of which rests on the wall of the capsule. 



Bnime 



23 



E. Changes In the nuclear cup* 

When solutions with a lower osmotic concentration than 
sea-water come in contact with the nuclear or protoplasmic cup 
it becomes thicker and the pseudopodia &re withdrawn so that 
the outline of the sperm, viewed from the top or bottom of 
the cup, is circular instead of star-shaped. The disappear- 
ance of the pseudopodia proceeds by a swelling at the base 
while the outer portion tapers very gradually to an extremely 
fine point. Compare Fig* 62 with 63 and 65. As the base 
widens out still father the rays are reduced to a very fine 
thread which either breaks off or is contracted into the body. 
When the pseudopodia break loose from their attachment the 
whole sperm is apt to move suddenly and then be borne away if 
there are any currents in the containing fluid. This sudden 
movement probably results from some of the pseudopodia break- 
ing loose slightly before the others. This rather than the 
explosion of the capsule may be the explanation of the "spring- 
ing of the sperm" discussed by Kolteoff '06. This rounding 
up of the protoplasmic portion of the sperm is apt to be com- 
pleted before any change takes place In the capsule. Some- 
times however the capsule may be completely changed before 
the disappearance of the pseudopodia. Probably In rapid ex- 
plosion the two take place simultaneously. 

8. Changes in the capsule . 

For the Interpretation of the entrance of the sperm into 
the egg the transformation of the capsule is much more import- 



24 



ant than the changes in the protoplasmic cup. We shall there- 
fore follow the capsular changes very carefully. The first 
change is the out pushing of the outer cavity of the inner 
tubule. CoBjpare Pig. 61 with 67. Here it is evident that 
the wall of the outer cavity of the inner tuhule has been 
everted, while the wall of the inner cavity (Pig. 61 » i.t.o.) 
has been stretched. It is difficult to see Just what change 
has taken place at this time in the central body. In some 
instances it appeared that it had been lengthed, and in some 
specimens I thought the end of it could be seen at the summit 
of the out pushed portion. It may be that the lengthening 
of this body is the force that turns this distal cavity inside 
out. 

In the next step of the capsular inversion the thick cov- 
ering of the out pushed part shown in Pig. 67 becomes turned 
out lateraly so as to form a collar (Pig. 68, r.) and the inner 
tubule becomes father everted. The collar formed at this 
stage persists unchanged throughout all the further modifica- 
tions of the capsule. The central body may now become great- 
ly increased in length so that it projects beyond the out 
turned part of the inner tubule (Pig. 69, c.b. also Pigs. 
70 to 72). Prom this stage on to the completion of the ever- 
sion there is little further Increase in the length of this 
axis. The everted portion of the inner tubule however swells 
out more and more (Pigs. 77 to 79). The transition from the 
condition shown in Pigs. 71 and 76 to that in Pigs. 77 to 79 
is brought about by a further everslon of the inner tubule. 
The part of the inner tubule involved in this second definite 



25 



everslon Is probably marked by the funnel-Bhaped portion In 
Pig. 76. The portion of the everted wall, derived from the 
part of the tubnle turned by this second everslon is Indicat- 
ed by the granule g. Pig. 77. At this stage there Is another 
pause while the out turned part continues to swell. 

Finally the tension becomes so great that another portion 
of the inner tubule is everted and as it turns the wall of the 
capsule is also turned through the collar formed in the early 
stage of the process of everslon. This last everslon is 
shown halfway completed in Pig. 80, and the completed process 
In Pig. 81. In the latter figure the central body stands on 
the apex of the everslon and the inverted capsule inv.c. is 
above the collar r. In dilute solutions of the salts used, 
the protoplasmic portion which contains the nucleus and mi- 
tochrondrlfld substance swells up to a spherical body as shown 
in Pig. 82. Often one finds on the slides, bodies like the 
one represented in Pig. 83. It is evident that these are ex- 
ploded sperms from which the everted inner tubule has dis- 
appeared leaving the central body, c.b.; the inverted capsule, 
Inv.v. ; the collar, r; and the shrunken nuclear cup, n.c. 

4. Changes i n the central body. 

We shall now return to a more complete consideration of 
the behavior of the central body and the part that it plays 
in the explosion of the sperm. These sperms are so very small 
it is difficult in many cases to distinguish the central body, 
especially in the live unstained material. Some significant 
facts however have been observed. As is shown in Pig. 61, 



26 



the central body Is oomposed of two distinct parts, the distal 
part within the cavity of the inner tuhule and a proximal part 
connecting the inner end of the tubule with the wall of the 
capsule. Whether the central body projects Into the outer 
cavity of the inner tubule or ends against the shelf separat- 
ing the inner and outer cavities of the tubule was not definite- 
ly determined but the latter seems to be the case. 

In Pig. 66, which was drawn from a sperm in the tubule 
of the testis that was fixed in Worcester's fluid and stained 
in iron haematoxylin, the central body projects through the 
apex of the capsule. This condition may have been brought 
about by an elongation of the central body or by a shrinking 
of the capsule. In either case it indicates that the central 
body is more or less rigid. One should notice also that the 
fixing fluid has caused a shrinking of the nuclear cup so that 
it is now more like a saucer than a cup. 

In Pigs. 69 to 72 which were drawn from living sperms the 
central body projects beyond the everted tubule like a rigid 
rod and one gets the impression that its elongation may have 
had something to do with the stretching of the tubule and the 
lengthening of that axis of the sperm. The idea that the 
central body is somewhat rigid is further supported by its ap- 
pearance in PigB, 73 and 74, where it stands out above the 
everted tubule. The same condition is produced in Pigs. 81 
and 82 » Probably the strongest evidence in favor of the rigid- 
ity of this structure is found in Pig. 75, where in lengthen- 
ing it has pushed backwards through the wall of the cep sule 
and pushed the nuclear cup away from the wall of the capsule. 



27 



There are some Indications that the central "body is not 
firm hut a plastlo, semiflnld euhstance. This Is supported hy 
the fact that It sometimes glides out through the Inner tubule 
at stages such as that shown In Pig. 76 and adheres to the 
surface of the everted tuhule In one or more amorphous masses 
(Pigs. 77,g.» 79 and 82). This condition may have heen brought 
about by a degeneration of the body as a result of keeping 
the sperms in the serum of the blood or in salt solutions. 
Sometimes in unexploded sperms the central body adheres to one 
side of the tubule instead of standing in the center, and it 
may be that it was only in such cases as this that it adhered 
to the everted wall of the tubule. 

5. Dynamics of Ever si on. 

We may now consider the forces Involved in the turning 
of the tubule and capsule inside out. We may divide this in- 
quiry Into two questions: (l) what are the external conditions 
necessary to initiate and csirry on the process? (2) What eire 
the internal conditions that respond to the external ones and 
determine the nature of the process? 

As stated above a decrease In the osmotic pressure of the 
medium in which the sperms lie, will oause the everslon. Un- 
exploded sperms taken from blood serum and placed in 5% KlOg 
do not explode; placed in 3% to A% ZSOg they take the forms 
shown in Pigs. 67 to 70; in 2?5 to 3^ KHO3 the forms in Pigs. 
70 to 72 and 77 are obtained; in 1.5^ to 1^ KNO3 the everslon 
proceeds to the stages shown in Pigs. 77 to 82. Like results 



28 



were obtained by treating the sperms with dilutions of 2.8^ 
HaCl or 4.25^ NaHOg. Hot all the individuals are equally af- 
fected by these solutions. Many of the sperms retain the un- 
exploded conditions of the capsule for a long time in a Z% 
MO3 solutions, and often none of them attain to the stage 
represented in ?lg. 82 when treated with 1% KMO3. 

Sperms kept for several days in 2.8?^ HaCl exploded when 
transferred to 4.25^ I^aHO^. Here we had an explosion when 
the sperms were transferred from a solution of one salt to 
that of another with equal osmotic pressure. Presh sperms 
do not explode when placed in 4.25^ SaHO^; therefore the 
sperms must have been changed by the HaCl, or the presence 
of these two salts must have had an effect that neither had 
when acting alone. To determine the factors here acting will 
require further experimentation. 

Some of the sperms explode whenever they are transferred 
to a slide and covered with a cover-glass. What may be the 
cause of such explosions was not determined. Koltzoff found 
that mechanical pressure would cause the explosion of the 
sperms of some decapods. I failed to produce any explosion 
by pressing on the cover-glass of a preparation containing 
them. Koltzoff '06 made extensive researches to find some 
specific stimulus that would cause a certain definite explosion 
which he believed to be the normal one but failed to find 
such a stimulus. It appears however that a careful investi- 
gation of the conditions which initiate the process followed 
up by an analysis of the conditions which may Increase the 



'.VHJ 



29 



pressure within the capsular cavity (Fig. 61 c.o.) would throw 
valuable light on this subject. My researches have been con- 
cerned with the exact changes that occur in the sperm rather 
than with the conditions that cause the changes. 

The second question, that Is the one concerning the in- 
ternal conditions which determine the response of the sperm 
to the external conditions, may now be considered. What is 
there In the sperm that may react to a decrease of the osmotic 
pressure of the solution which surrotmds it? An examination 
of Figs. 68 to 82 clearly shows that it is the capsular cavity 
that increases in size. It must therefore contain a substance 
which is Isotonic with sea-water and with the blood of the 
crab and which absorbs water when placed in any solution which 
is of a lower concentration. This water is doubtless taken 
in through the wall of the inner tubule which seems to be semi- 
permeable, while the outer wall of the capsule is probably 
impervious. 

Another striking feature of the explosion is the remark- 
able extensibility of the wall of the inner tubule which is 
everted to form the wall of a structure many times larger 
than the capsule. The central body must also be considered 
as one of the structures taking a part in the explosion of the 
capsule. We have therefore three changing structures, a swell- 
ing mass, a stretching membrane, and an elOEgating body, each 
of which take a part in determining the form of the Inversion. 
To these must be added two structures which do not change and 
are resistant in their nature. These are the wall of the 



ntti-} 



.Trrt-M'jtT 



30 



capsule and the collar surrounding the hole In the capsule, 
through which the tubule Is everted. Let us now follow the 
interaction of the forces Involved in the behavior of these 
changing structures. For this purpose we shall divide the 
explosion into four stages. 

Stage 1. The eversion of the outer cavity of the tuhule 
(Pig. 67). Two forces probably take part in this, the pres- 
sure in the capsular cavity and the elongation of the central 
body. 

Stage 2. The elongation of the everted outer cavity (Pigs. 
68 to 71 and 76). This results in the formation of the collar 
(Pig. 68,r.). Here again two forces may be involved, the 
swelling of the material in the capsular cavity and the fur- 
ther elongation of the central body which stretches the por- 
tion of the inner tubule which bounds the inner tubular cav- 
ity. The fact that the everted portion is sometimes longer 
in the axis through which the central body passes indicates 
that this body may be exerting an out-pushing force. If this 
is the case, we have here an elastic body that has become 
active by being released from compression, that is the central 
body elongates like a coiled spring. This action is fully 
discussed by Koltzoff . The pressure in the capsular cavity 
is sometimes showa by the squeezing of the central body out 
through the outer end of the tubule when it has lost its re- 
sistant properties. 

Stage 3 . The second eversion of the inner tubule (Pigs. 
77 to 79). Prom the condition shown in Pigs. 71 and 76, the 
increasing pressure in the capsular cavity causes the wall 



31 



of the everted tutule to swell up to the form shown in Pig. 72. 
Finally the pressure becomes so great that the ring which 
formed the division "between the inner and outer tuhular cavi- 
ties (Fig. 61, i.t.c. and o.c.) gives away and a part of the 
tubule bounding the inner tubular cavity becomes everted. 
Portions of the central body often adhere to the wall of the 
tubule and are carried outward and so mark the extent of this 
second eversion (Fig. 77, g. ) . 

Stage 4« The third eversion of the tubule , accompanied 
by the inversion of the capsule (Figs. 80 to 82). The in- 
ternal pressure continues to increase as is shown by the 
bulging out of the walls of the everted portion (Fips. 78 and 
79). This brings a strain upon the axis in which the tubule 
and central body lie. This tends to stretch these structures 
as is shown by the incurving of the apical wall of the everted 
portion in Figs. 78 and 79. This causes the base of the evert- 
ed part to press on the sides of the capsule. This pressure 
on the sides of the capsule together with an up-pulling along 
the line of the central body results in turning the capsule 
through the collar when the last section of the tubule is 
everted. In Fig. 82 the portions of the everted wall con- 
tributed by the second, thlrci and fourth stages of the ever- 
sion are probably Indicated by the granules g^ and g^. Thus 
we see that the whole transformation may be explained by the 
increase of pressure in the capsular cavity together with 
tensions along the line of the inner tubule and the central 
body*. 



T«C 



32 



Efforts were made to reverse this process ty placing the 
sperms In very concentrated solutions of the salts used. The 
only effect of this treatment was a shrlnkihg of the everted 
portion, which would again swell up and the process of eversion 
continue when dilute solutions were again used. After the 
explosion had reached the stage presented in Pig. 8E the only 
part affected by concentrated solutions was the protoplasmic 
portion at the bottom. It is also true that this is the only 
part that takes methylene blue, methyl green or thronin 
stains when these are applied to the living sperms. It was 
rather surprising that the contents of the everted capsule 
were not stained by these stains. Sometimes a few granules 
can be seen in this cavity. 

Figures 73, 74, 84 and 85 were made from sperms that had 
been kept in a 5% KUO3 solution for 51 days. We observe here 
that partial explosion had taken place. Those shown in Figs. 
84 and 85 had reached the stage corresponding to Fig. 70. 
When these were treated with a solution of lower concentration 
the eversion continued but the wall of the part already evert- 
ed seemed to be hardened so that it made an elongated collar 
through which the further inversion took place. 

6. The effect of reagents on the everted sperms. 

After trying several fixing reagents it was found that 
for imbedding and cutting the crab's eggs Morgan's fluid gave 
decidedly the best results. This fluid is a saturated solu- 
tion of picric acid in ^0% alcohol, to 100 c.c. of which 2 o.c. 
of H2SO4 are added. Sperms in various stages of the process 



;jiii:nf tsdcfT' 



33 



of inversion and those In the normal mature condition were 
motinted under the microsoope and killed with this fluid in 
order to determine its effect upon them. A considerable 
amount of shrinking was observed, hut no decided change in 
the relationship of the parts seemed to take place. Figures 
86 and 87 were drawn from sperms exploded in distilled water, 
fixed in Morgan's fluid, stained in thionin and eosin, dehy- 
drated In alcohols, cleared in xylol and mounted in balsam. 
The general relations of the parts is the same as in Figs. 81 
and 82. The space between the everted tubule and the invert- 
ed capsule seems to have shrunk relatively more than the in- 
verted capsule. Having now observed the behavior of the 
sperms under experimental conditions we may proceed to our 
observations concerning the entrance of the sperm and the 
process of fertilization. 

711. THE ENTRAHCE OF THE SPERM. 

Eggs were taken from the lumen of the ovary just after 
the crab began to spawn, and were fixed in Morgan's fluid, 
imbedded in paraffin and sectioned. A microscopic examina- 
tion of these eggs showed the sperm in the act of entering 
the egg. The best stain for the study of these sections is 
thionin, for it stains the chromosomes in the mitotic figure 
of the nucleus of the egg and the nuclear cup of the sperm a 
deep blue. It stains the everted portion of the sperm faint- 
ly and the food material, cytoplasm and egg-shell are unstain- 
ed or only faintly stained. This treatment makes it possible 



34 



to find these minute structures in the relatively immense egg. 
After one has heGorne familiar with these structures and their 
position on the egg it is possible to find them quite readily 
with other stains such as Delafield's haematoxylin, iron- 
haematoxylin, or safranin and Liohtgrun. 

The relation of the sperms to eggs taken from the lumen 
of the ovary is shown in Pigs. 88 to 92. Sperms in the same 
condition are also found In eggs taken from the oviduct as 
is shown in Pigs. 9Z and 94. How when these figures are com- 
pared with Pigs. 96 and 87, which have been treated with the 
same fixing reagents, it is evident that it is the everted 
portion of the sperm that has gone through the shell of the 
egg. The nuclear cup (n.c. Pigs. 91 and 92) is on the out- 
side of the shell. The everted tuhule forms a vesicle with- 
in which one sees the Inverted capsule (inv.c. Pig. 91). Hera- 
after I shall call this everted tuhule and capsule, the sperm- 
vesicle. At the inner-end of this sperm-vesicle the ejected 
central hody may he seen (Pigs. 88, 91 to 94 c.b.). That the 
part which remains outside is the nuclear cup with its radiat- 
ing pseudopodla can he more clearly seen hy a surface view of 
the structure as it lies upon the eg^. Such a view is present- 
ed in Pig. 95. Furthermore the staining reactions are in ac- 
cord with those observed in the mat:ire sperm and the artificial- 
ly exploded ones and are as follows: 

) the part outside of the shell-blue. 
Thionin and eosin ( 

) the part inside of the shell--red. 

„ (part outside of shell--red 
Safranin and Liohtgrun ) 

(part inside, green mixed with red. 



35 



( part outside of shell, tlack. 
Iron-Haematoxylin ) 

( part inside, 'brown except the central "body 
) which is hlaok. 

Koltzoff '06 claimed that the sperms. In certain decapods 
settled on the egg with the nuclear cup towards the egg and the 
capsule pointed away from the egg. He was also of the opinion 
that the rehound from the explosion of the capsule was suffi- 
cient to drive the nucleus Into the egg. On eggs taken from 
the ovary of Menippe mercenaria, I found a few sperms attach- 
ed as shown in Fig. 96 with the nuclear cup next to the shell 
of the egg. That the eversion of the capsule does not force 
the nucleus through the shell in this case is shown in Pig. 
97, where a sperm has exploded with the nuclear cup against 
the egg. So far as my ohservations go there is no evidence 
whatever that the eversion of the capsule causes any sudden 
movement of the sperm hody as a whole. 

The niimber of sperms that have pierced the shells of the 
eggs is much greater for eggs taken from the oviduct than for 
those taken from the lumen of the ovary. The ntimher was count- 
ed In a few eggs that had Just heen spawned and the number 
per egg was as follows: 28,44,52,52,54, 71, 73 and in one ex- 
ceptional case 679. 

So far there seems to "be no doubt as to the behavior of 
the sperm in entering the egg, but we may ask; Is this the 
final stage in the entrance of the sperm? Is not the nuclear 
cup drawn through the shell at a later stage? If it is not 
what becomes of it? 

That the nuclear cup does not enter the egg^ but falls 
off is shown in Figs. 98 to 101. Here we see that the nuclear 



.3,^$.:^ 



36 



cup has moved away from the egg-shell and that a strauid of sorae 
substance hy which It was prohably attached to the hotton of 
the capsule is drawn out with it. Sometimes the nuclear cup 
breaks loose from the strand and leaves it projecting through 
the shell into the capsule (Pig. 100). In eggs taken from 
the oviduct or from the pleopeds Just after spawning, large 
numbers of sperm-vesicles are found sticking to the inside 
of the shells after the nuclear cup has fallen off (Pig. 101). 
It is very clear then that in most cases at least the nuclear 
cup does not enter the egg* But, does it thus fall off from 
the particular sperm that fertilizes the egg^ or only from 
those which have failed to perform the work of fertilization? 
This question can best be answered by a further study of the 
events of fertilization. 

VIII. PERTILIZATIOBT. 

In eggs taken from the oviduct or just after leaving it, 
many sperm-vesicles may be found lying on the edge of the 
cytoplasm as shown in Pigs. 102 and 103, while one is found 
down in the cytoplasm (Pigs. 104-108). Here it is evident 
that the movement of the cytoplasm has carried the vesicle 
below the surface. Before the sperm-vesicle enters there is 
a layer of cytoplasm Just inside of the egg-shell. The rest 
of the egg is filled with spherules of food material in the 
interspaces of which the cytoplasm extends from the peripheral 
layer, by fine strands, all through the egg. The fact, that 
the sperules of food move apart and a small mass of cytoplasm 



:c? 



37 



acoompanles the sperm-vesicle into the egg» Is "best explained 
"by supposing that the first vesicle that comes in contact 
with the cytoplasm initiates a flowing movement of the latter 
along the inner surface of the shell, from all sides towards 
the newly entered vesicle. The cytoplasm moving thus along 
the inner surface of the shell towards one point would he de- 
flected in towards the center of the Qggt and would tend to 
carry the vesicle in with it. We may suppose further that 
when the cytoplasm has once responded to such a stimulus its 
physiological state is so changed that it will not respond 
to another. As a result only one vesicle becomes imbedded 
In the cytoplasm of the egg where it is to be treuasformed in- 
to the male pronucleus. 

The vitelline membrane (Pig. 104, v.) is formed just after 
the entrance of the sperm-vesicle into the cytoplasm and 
the vesicles that failed to enter lie between it and the 
shell of the egg» The first polar body (Pig. 114) which is 
cast off while the eggs are passing through the oviduct is 
also found between the vitelline membreme and the shell. 

The first step in the transformation of the sperm-vesi- 
cle into the male pronucleus is a thickening of its lateral 
walls. This may be observed in Pigs, 105 to 111. Accompany- 
ing this there is an increase of affinity for the stains used 
(thionln and Delafield's haematoxylin) . Uext there seams to 
be an extrusion of the old capsular wall which, if we recall 
the method of the eversion of the sperm, we know, forms the 
inner lining of the sperm-vesicle. The discarding of this 
capsular wall was not clearly made out, bnt Pigs. 109 and 110 



38 



indicate that suoh a change takes place. In eggs taken soon 
after spwaning the sperm-vesicle has increased considerably 
in size and the wall has taken on a vesicular appearance. 
This is shown in Figs. 110 eoid 111 which show the sperm-ve si- 
de in two different aspects. The cavity of the vesicle 
gradually disappears leaving only a notch on one side (Pigs. 
108, 109, 110 and 112). Figure 112 was about an hour old. 
In eggs two hours old the vesicle has taken on the appearance 
of an ordinary nucleus containing granules of chromatin (Fig. 
116). At this time it has gone about one fourth of the dis- 
tance from the circumference to the center of the egg (Fig. 117) 

We will now turn our attention to the egg-nucleus. When 
the eggs are set free in the lumen of the ovary Just before 
spawning begins, the spindle for the first maturation division 
is already formed and its long axis is parallel with the sur- 
face of the egg (Fig. 113). As the egg passes through the 
oviduct the spindle turns to a position perpendicular to the 
surface of the egg and the first polar body is cut off. This 
is shown in Fig. 114 which is from em egg that has Just pass- 
ed out of the oviduct. Efforts were made to count the chrom- 
osomes just after this division. Twenty-five to twenty-eight 
were counted. Thus the double number would be fifty to fifty- 
six. They are however so small and placed so closely together 
that it is difficult to distinguish them accurately. 

Between one and one and a half hours after spawning the 
second maturation division takes place. The second polar 
body is apparently not cut off, but remains in the egg where 
it degenerates and is absorbed by the cytoplasm. 



39 
The female pronucleus Is now formed and proceeds to the 
center of the egg where It meets the male pronucleus. Figures 
116 and 116 are drawn from nuclei fixed at 2 hours and 15 min- 
utes after spawning. At this time it is not possible to tell 
which is the male and which the female pronucleus tmless the 
slightly concave side of the one shown in Pig. 116 indicates 
that it is related to the nucleus shown in Fig. 112. In this 
case it would he the male pronucleus. The nuclei have grown 
rapidly and continue to do so until they reach the center of 
the ogg. Their contents are finely granular. These granules 
increase in size as the nuclei hecome larger. Figures 118 and 
119 show the position of the nuclei four hours after spawning 
and Fig. 120 (from en agg six hours old) shows them lying side 
by side in the center of the egg. They have become elongated 
and many times larger than they were when first formed. From 
the above description of fertilization it is evident that the 
nuclear cup takes no part in the formation of the male pro- 
nucleus for the latter is derived from the sperm-vesicle which 
is the Inverted capsule. 

This completes the description of the structure and be- 
havior of the male cells in the stone-crabe. We have here some- 
thing unique in the method by which the sperm enters the egg 
and something exceptional in the phenomena of fertilization. 
These observations raise several theoretical questions, some 
of which we will now briefly consider. 

IX. Discussion. 
We have here a case in which an Infolded vacuole that 
arose in the cytoplasm is everted through the shell of the 
egg and fertilizes it. How may such an event be brought into 



40 



harmony with the existing theories ooncernlng the chromoaomes? 
In all other cases of fertilization the nucleus with its 
chromosomes or at least Its chromatin is considered the essen- 
tial thing; the bearer of the paternal qualities to the egg. 
The part that they play in the theories concerning heredity 
is too Important and useful to be lightly discarded. But, 
granting all that is claimed for the chromosomes, we are never- 
theless face to face with the fact that in most cases they 
disappear during the telophase and are reformed in the next 
prophase of cell division. Between the miotic divisions they 
can be followed from one spindle to the next and in some other 
oases some Investigators have claimed to have been able to ob- 
serve the continuity of Individual chromosomes from one divi- 
sion of the cell to the next one. But these are exceptions. 
The problem of the origin of the chromosome is a real problem. 
For many reasons, in our final analysis, we must go back of 
the chromosome. So without attacking the proposition that 
chromosomes are the means for distributing the hereditary 
elements at the time of division, we may take up the question 
of the origin of the chromosome before division. 

The phenomena described in this paper force us to con- 
sider this question if we are to bring the facts concerning 
fertilization in this crab, in line with existing theories. 
Does any of the chromatin from the nucleus of the spermatid 
enter the egg"? We have shown that it is the substance in 
the wall or in the cavity of the capsule that enters and 
fertilizes the egg' Now is there any evidence that the chrom- 
atic substance in the nucleus is transferred to the capsule 



snT 



ei-ff 



41 



during spermatogenesis f 

Gro'b'ben '78 claimed that the capsule Is derived from the 
nucleus of the spermatid. He described a change In the con- 
sistency and a reduction in size of the nucleus which occurred 
simultaneously with the development of the capsule. He seem- 
ed to he of the opinion that the nuclear material was trans- 
ferred hy diffusion from the nucleus to the capsule. 

Herrmann '90 suggests that when one follows the parallel 
transformations of the capsule and the nucleus, one gets the 
impression that there Is a sort of migration of the chromatic 
suhstance from the nucleus to the capsule. 

Brandos '97 found two substances in the nucleus of the 
spermatid. One was stained blue with methylene blue, the 
other red with acid fuchsln. The latter settles to one side 
of the nucleus and then passes out into the cytoplasm. 

The later workers Koltzoff '06 and Spltschakoff '09 de- 
*^ scribe no such d process. 

In my own investigations I have noted a decrease in af- 
finity for chromatic stains, and in the size of the nucleus. 
The capsule on the other hand showed an increasing affinity 
for iron-haematoxylin and safranin. These facts suggest a 
transfer of nuclear material. 

Finally if we may accept the views of Stauffacher '10 
and Dersohan 'll, that basichromatin is derived from oxy- 
chromatin, the former being deposited from the latter, we may 
postulate a theory for the explanation of the phenomena of 
fertilization in this crab. I do not claim that the facts 



42 



establish the theory. They only suggest it. Some of the 
"b as i chromatin in the nucleus of the spermatid is dissolved 
by the oxychrooatin and transferred to the capsule. After 
the capsule is everted into the egg and has entered the cyto- 
plasm of the latter, the b^sichromatin is redeposited and 
thus the granular structure of the male pronucleus appears. 
It may be possible to explain the number of chromosomes that 
appear by supposing that there are a certain number of dif- 
ferent kinds of molecules which are deposited out of the 
oxyohromatin and that these have such an affinity for each 
other that they are aggregated into a definite number of 
groups or they may be of such a structural nature than they 
can fall only into certain groups. Of course, I claim for 
this only that it Is a possible explanation of phenomena 
which are apparently not In accord with the conception of an 
individual continuity of the chromosomes. 

Z. SUMMARY. 

1. The seminal elements in Menlppe mercenaria arise 
from a single row of primary spermatagonlal cells which per- 
sist along one side of the testicular tubule. 

2. The tubule is divided into three or four regions by 
longitudinal partitions composed of epithelial cells. The 
seminal elements in the division next to the row of spermato- 
gonlal cells, are younger than those in any other division. 
The region on the opposite side contains mature sperms. The 
seminal elements In one end of a given division are further 



43 



along in their development than those In the other end. 

g. The spermatogonia! nuclei lie in a oommon cytoplasmic 
mass and multiply irregularly without the formation of a 
spireme. A spireme and synapsis occur in connection with the 
first miotic division. The second miotic division follows 
soon after the first. 

4. In the mature sperm, the protoplasmic portion, con- 
taining the nucleus, is cup- shaped. Prom the rim of the cup 
peeudopodia project out like the rays of a star. There Is 
a capsule half imhedded in the cup. An inturned tubule is 
connected with an opening in the distal portion of the cap- 
sular wall and a rod-like central hody arises from the prox- 
imal side of the capsule and projects into the inner tuhule. 

5. In the transformation of the spermatid, the nucleus 
becomes uniform in consistency, reduced in size and cup-shaped. 
A mitochrondrial ring is formed between the nucleus and the 
capsule. The capsule arises as a vacuole in the cytoplasm. In 
the course of its development it shows an increasing affinity 
for nuclear steins. 

6. The central body develops from a granule that appears 
on the proximal side of the capsule. The inner tubule is 
formed from two vesicles that arise at the distal end of the 
central body. 

7. Hypotonic solutions of various salts and possibly 
other stimuli cause a lengthening of the central body, an 
eversion of the inner tubule and an inversion of the wall 
of the capsule. 

8. When the sperms come in contact with the egg under 



?er t-Xi 



44 



normal conditions, the capsule is usually applied to the shell 
of the egg and the nuclear cup is directed away from the egg. 
In this position eversion takes place and the ejected central 
hody, the inner tubule and the capsule with its contents are 
thus turned through the shell into the egg. 

9. The nuclear cup is left on the outside of the egg 
and it soon falls off. 

10. The wall of the capsule together with its everted 
contents, which we now call the sperm- vesicle, sinks into 
the cytoplams of the egg^ where it is enlarged and transform- 
ed into the male pronucleus. 

11. The contents of the capsule may he derived from 
the nucleus of the spermatid and is prohahly oxychromatin 
which deposits hasichromatin after it enters the egg and so 
gives rise to the chromosomes in the male pronucleus. 



(TITO 



45 



LITERATURE CITED. 

Brandes, G. '97, Die Spermatozoen der Dekapoden. Sitzungsbe- 
richte d.K.P. Akad. d. Wlssen, Berlin, 1R97. 

II 
Derschan, M. '11, ITber KermbruGken nnd Kernsubstance in pflanz- 

lichen Zellen. Arch. f. Zellforseh. Bd.7. 

Gllson, Gr. '86, Etude comparee de la spermatogenese chez les 
arthropodes. La Cellule, Tome II. 

Grobben, C. '78, Baitrage zur Keuntniss der mazmllchen 

Geschlechtsorgane der Dekapoden. Arbeit. a. d. 
Zool. Inst. d. Univ. Wien. Bd. i. 

Herrmann, G.'90, Botes sur la structure et le developpement 
des spermatozoides chez les Decapodes. Bull. 
Sc. de la Prance et de la Belgique. Vol. XZII. 

Koltzoff, U.K. '06, Studlen uber die Gestalt der Zelle . I. Un- 

tersuchimgen uber die Spermlen der Decapod eh, 
als Einleitung das Problem der Zellengestalt. 
Arch.f. mikros. Anat. Bd. 67. 

Sabatler, A. '93, De la Spermatogenese chez les Crustaces 
Decapodes. Travaux d.l'Inst d. Zool. de 
Montpellier et Sta. ilar. Gette. 3er.9. Mem. 
No. 3. 

Spitschakoff , Th. Spermohistogenese bei Gariden. Archiv.f . 
Zellforschung-. III. 

Stanffacher, Hch. Beitrage zur Zenntniss der Zernstrukturen. 
Zeitschrift f. wissench. Zoologue XGV. Bd. 



46 



BXPLAUATION OP THK i<'IGURES. 



Fig. 1. Transverse section of a testicular tubule of a 
small crat. Fixed In Petrunkewitsch* s fluid. Z600 Spermatogonia 
at the top* Sperms at the "bottom, spermatids between. 

Fig. 2. Transverse section of testicular tubule, p.s., 
primary spermatogonlal cell. The mass of spermatogonia larger 
than in Pig. 1. 

Fig. 3. Transverse section of testicular tubule showing 
larger mass of spermatogonia, p.s., primary spermatogonlal 
cell. 

Fig. 4. Transverse section of testicular tubule showing 
a large mass of spermatocytes in synapsis, p.s., primary 
spermatogonlal cell. 

Figures 5-llS (except 7u-72) were all drawn with the 
camera lucida and a Zeiss 1.5 m.m. apochromatic objective and 
a compensating ocular (either a No. 6 or XJo. 8). Then the 
drawings were enlarged so that in the plates there is a mag- 
nification of 3000 diameters. In making Pigs. 7u-72 the 
camera lucida was not used. 

Fig. 5. Resting phase of a spermatogonlal nucleus. 

Fig. 6« Prophase of a spermatogonlal nucleus showing 
the paired chromosomes which were seen in the upper one-half 
of the nucleus. 

F ig. 7. Optical section df a spermatogonlal nucleus 
showing the peripheral arrangement of the paired chromosomes 
in the prophase. 

Fig. 8» The mitotic figure in a spermatogonlal division. 

Fig. 9. Early prophase of the first miotic division of 
the spermatocyte . 

Fig. 10. SytapslB in the first miotic division of a 
spermatocyte. 

JTlgs. 11 and 12. The stage following synapsis showing 
the spireme loosened up and separating into chromosomes. 

Fig. 13. First miotic division, stage Jxlst preceding 
the formation of the equatorial plate. 

Fig. 14. Equatorial plate and spindle in first miotic 
division. 

Fig. 15 . A tripolar division of the nucleus of a sperma- 



47 



tocyte of the first order. 15a is a drawing of the same 
nucleus made from a lower plain and showing a portion e. of 
the chromatin which was not included in the equatorial plate. 

Pig. 16. Chromosomes found In the equatorial plate of 
the first miotic division. 

Figs. 16a and 16h. Portions of the first miotic figure 
in the metaphase, showing the chromosomes. 

Figs. 17 and 18. Two stages in the anaphase of the 
first miotsis. 

Figs. 19-22. Show different stages and variations in the 
early telophase of the first miotic division. Interzonal 
flhers are shown stretching hetween the masses of chromatin 
and the mld-hody apparently forming a hand around the flhers. 

Figs. 23-25. The second miotic division: Stages in the 
formation of the equatorial plate. 

Fig. 26. The metaphase of the second miotic division. 

Fig. 27. Telophase of second miotic division, showing 
Intersonal flhers and the mid-body. 

Fig. 28. Later telophase. The centrosome is still vis- 
ible here and is still attached hy flhers to the nucleus. A 
clear area also surrounds the nucleus. 

gigs. 29-52. Different stages In the formation of a 
reticulate nucleus in the spermatid. A black granule, the 
centrosome may be seen and a more or less complete zone free 
from granules around the nucleus. 

Figs. 55-56. Spermatids with vacuoles in the cytoplasm, 
c.a. mass of finely granular cytoplasm which may be the mlto- 
chrondria. 

Fig. 57. Spermatid with vacuole or capsule and nucleus. 
The periphery of nucleus more densely stained than center. 

Fig. 58 . Spermatid showing a small clear vacuole in the 
nucleus. 

Figs. 59-42. Later stages in the transformation of the 
spermatids mt. mitochondrial substance. 

Pig. 45. Spermatid showing two sides of a dark ring, 
d. in the capsule; mt. mitochondria, also the central body 
on the nucleus at the bottom of the capsule. 

Fig. 44. Spermatid showing two granules on the border 
line between the nucleus and capsule. 



48 



Figa. 45-47 » Spermatid from different view points show- 
ing the mitochondria, mt. and a granule. In "bottom of cap- 
sule. 

Plg» 48« Spermatid with mitochondrial ring completed. 

Pigs. 49-60. Spermatids showing stages In the develop- 
ment of the central "body and the Inner tuhule within the cap- 
sule. A clear space appears for a time "between the capsule 
and the nucleus. 

Pig. 61. Mature sperm, fixed In vapor of osmic acid and 
stained In gold chloride preparation, n.c, nucleus cup; p.s., 
pseudopodla; c, wall of the capsule; c.c, cavity of the 
capsule; l.t., inner tubule; l.t.c. Inner cavity of the 
Inner tuhule; c.b., central body; o.c. outer cavity of the 
inner tubule. 

Pig. 52. Sperm viewed from the top, mounted In 4% M0„. 

Fig. 63. Sperm mounted In the serum of the crab's blood. 

Pig. 64. Side view of sperm In the serum of the blood. 

Pig. 65 . Sperm in serum with pseudopodla all turned to 
one side by currents In the serum. 

Pig. 66. Sperm in testicular tubule. Fixed in Worcester's 
fluid. Stained In Iron-haematoxylln, central body projecting 
from the top. 

Pig. 67. A sperm treated with 3% EIO.'^, fixed with Morgan's 
fluid and stained with Delafleld's haematoxylln. 

Fig. 68. A sperm treated with 3% KtlOs, fixed with Mor- 
gan's fluid and stained with eosln. 

Figs. 69-85 . Were all drawn from living sperms that had 
been treated with hjrpotonie solutions of salts, and show 
various stages in the eversion of the capsule, r.. the collar; 
c.b., central body; Inv.c. Inverted capsule; g, g-^ and g^, 
pieces of the central body on the everted wall of the inner 
tubule. 

Figs. 86-87 . Sperms that were exploded in distilled water 
and fixed in Morgan's fluid then stained with thlonln and 
eosln. 

Figs. 88 to 92 . F.verted sperms extending through the shell 
of the egg, from the lumen of the ovary; fixed in Morgan's 
fluid. In Fig. 89 the shell of the egg Is badly warped, c.b. 
ejected central body; Inv.c, Inverted capsule; n.c, nuclear 
cup; r., collar of the capsule. 



49 



Figs. 93 and 94 » Everted sperms in the shells of eggs from 
the oviduct, c.h., central hody ejected; r. , collar. 

Fig. 95. The portion of the sperm v/hlch remains on the 
outside of the egg seen from the hottom of the nuclear cup. 

Pig. 96. Sperm on the shell of the egg with nuclear cup 
next to the shell. 

Pig. 97. A sperm that has exploded with nucleus next to 
the egg. 

Figs. 98-111. Portions of eggs with sperms, taken from 
the oviduct or soon after leaving It. 

Figs. 98 and 99 . Show the nuclear cup falling away from 
the egg and pulling a strand of some suhstance out with it. 

Fig. 100. Sperm-vesicle with the strand from which the 
nuclear cup has hroken away, projecting through the shell 
into the vesicle. 

Pig. 101. Sperm-vesicle just inside the shell. 

Pigs. 102 and 103. Sperm-vesicles lying on the cytoplasm. 

Fig. 104. Sperm-vesicle that has just entered the cyto- 
plasm, v., vitelline memhrane. 

Pigs. 105-108. Sperm-vesicles down in the cytoplasm. 

Pig. 109. Sperm-vesicle with capsular wall projecting 
out of it. 

Pig. 110. Sperm-vesicle seen from one side; shows vesi- 
cular structure. 

Pig. 111. Sperm-vesicle seen from top-vesicular struc- 
ture. 

Pig. 112. Sperm-vesicle in egg 1 hour, 15 minutes old. 
This may now "be called the male pronucleus. 

Pig. 113. 'Rgg from the lumen of the ovary showing the 
spindle of the first miotic division. 



Fig. 114. First polar body of an egg Just spawned. 

Pigs. 115 and 116. Two pronuclei found in one egg Z 
\ and 15 minutes old. 

Fig. 117. Egg 2 hours 15 minutes old, h., transverse sec- 



50 



tlon of a hair of the pleopod with the shell of the egg wrap- 
ed part of the way around it; n., pronucleus, prohahly the 
male; v., vitelline memhrane. X425. 

Figs. 118 and 119 » Egg 4 hours old showing the pronuclei. 

Fig« 120 . Egg 6 hours old showing the pronuclei side hy 
side in the center of the egg. 



IlKi 



121. Diagram of the ovary, the seminal receptacle 
The latter turned out to one side so as to 



and oviduct. 

hring it in the same plane with the rest of the ovary; c, 
portion of the seminal receptacle lined with chitin; g., 
glandular portion of the seminal receptacle; o., opening 
between the glandular and chitinous portion of the recept- 
acle; od., oviduct. 

Fig. 122. Seminal receptacle at the time of Molting, 
c, chitinous portion; g. , glandular portion. 



51 



VITA. 

I was Ijorn near Carthage, Rush County, Indiana, July 
15, 1676, and am the son of Josiah and Margaret F. Binford. 
My family moved to western Kansas in 1885. I graduated from 
the Haviland Academy In 1896. Then I taught a district 
school one year, studied at warlham College, Richmond, Ind. 
two years, and again taught a district school at Haviland, 
Kansas for one year and the next year returned to Sarlham 
College where I received the B.3. degree in 1901. Since 
1901 I have held the position of Professor of Biology at 
Guilford College, U.C. During the summer quarters of 19u2 
and 1903, and for five quarters in 1905- '06 I studied in the 
University of Chicago, devoting my attention principally to 
the subject of botany, and received the degree of S.M. from 
that institution. 

During the academic year of 1907- '08 I devoted my time 
to zoological studies at Johns Hopkins University. I was 
scientific assistant at the U.S. Marine Biological Labora- 
tory at Beaufort, H.G. during the four summers; 1908, '09, '10 
and '11. At this laboratory I worked on the life histories 
of several crabs, though primarily investigating that of 
Menippe raercenaria. 

In the fall of 1910 I returned to the Johns Hopkins Uni- 
versity where the past two years have been spent in working 
up the foregoing dissertation and in other zoological studies. 

Raymond Binford. 
Baltimore, Md. May 1, 1912. 



i*».; 









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